恩诺沙星在鸡肉组织中残留的ELISA检测方法研究

Establishment of an indirect competitive enzyme-linked immunosorbent assay for the residues of enrofloxacin in chicken tissue

分别用N-羟基琥珀酰亚胺法和氯甲酸异丁酯法把恩诺沙星（ENR）与载体蛋白牛血清白蛋白（BSA）和鸡卵清蛋白（OVA）偶联制备免疫抗原和包被抗原,免疫新西兰大白兔得到ENR的多克隆抗体,建立了ENR间接竞争ELISA方法。结果表明：抗ENR血清效价达达1∶212以上,得到标准曲线的线性回归方程为Y=-0.2341X＋0.1193（R2=0.9878）,中值（IC50）为36 ng/mL,最低检测限（LOD）为10 ng/mL,标准曲线的线性范围为10-1000 ng/mL。批内变异系数为3.18%-7.64%,批间变异系数为9.69%-11.94%,鸡组织中的ENR的回收率为76.5%-89.42%。本试验建立的ELISA方法能够满足恩诺沙星兽药残留检测要求。

Enrofloxacin （ENR） was coupled with the carrier protein bovine serum album （BSA） to prepare the immune antigen ENR- BSA by N-hydroxysuccinimide ester （NHS） method and with chicken ovalbumin （OVA） to prepare coating detection antigen by isobutyl chloroformate method. ENR-BSA as immunizing antigen was vaccinated to New Zealand albino rabbit in order to obtain the polyclonal anti- body to enrofloxacin. An enzyme-linked immunosorbent assay （ELISA） method was established. The result indicated that specificity antibody titer reached above 1：212. The regression equation, the 50% inhibition concentration （IC50）, the limits of detect （LOD） and the linear range about the standard curve of enrofloxacin were Y = -0. 234 1X ＋ 0. 119 3 （R2 = 0. 987 8 ） , 36ng/mL, 10ng/mL and 10 - 1 000ng/mL, respectively. The coefficients of variation for the Ci-ELISA were 3.18% - 7.64% in intra-assay and 9. 69% - 11. 94% in inter-assay. The recoveries in chicken ranged from 76. 5% to 89.42%. The established ELISA method will satisfy the determination for the residues of enrofloxacin in chicken.